Mutational screening of six genes in Chinese patients with congenital cataract and microcornea.

PURPOSE
To identify mutations in 6 genes of 9 Chinese families with congenital cataract and microcornea.


METHODS
Nine unrelated families with congenital cataract and microcornea were collected. Cycle sequencing was used to detect variants in the coding and adjacent regions of the crystallin alpha A (CRYAA), crystallin beta B1 (CRYBB1), crystallin beta A4 (CRYBA4), crystallin gamma C (CRYGC), crystallin gamma D (CRYGD), and gap junction protein alpha 8 (GJA8) genes.


RESULTS
Upon complete analysis of the 6 genes, three mutations in 2 genes were detected in 3 families, respectively. These mutations were not present in 96 normal controls. Of the three mutations, two novel heterozygous mutations in GJA8, c.136G>A (p.Gly46Arg) and c.116C>G (p.Thr39Arg), were found in two families with congenital cataract and microcornea. The rest one, a heterozygous c.34C>T (p.Arg12Cys) mutation in CRYAA, was identified in three patients from a family with nuclear cataract, microcornea with axial elongation. No mutation in the 6 genes was detected in the remaining 6 families.


CONCLUSIONS
Mutations in GJA8 and CRYAA were identified in three families with cataract and microcornea. Elongation of axial length accompanied with myopia was a novel phenotype in the family with the c.34C>T mutation in CRYAA. Our results expand the spectrum of GJA8 mutations as well as their associated phenotypes.

In this study, we performed mutational screening of 6 genes (CRYAA, CRYBB1, CRYBA4, CRYGC, CRYGD, and GJA8) in 9 Chinese families with congenital cataract and microcornea. Three mutations in GJA8 and CRYAA were identified in 3 families.

METHODS
Patients: Nine families with congenital cataract and microcornea were collected at the Pediatric and Genetic Eye Clinic of the Zhongshan Ophthalmic Center, Guangzhou, China. Written informed consent conforming to the tenets of the Declaration of Helsinki and following the Guidance of Sample Collection of Human Genetic Diseases (863-plan) by the Ministry of Public Health of China were obtained from the participating individuals or their guardians before the study. Congenital cataract represents cataract presented at birth or noticed in the first few months after birth. Microcornea represents a cornea with horizontal diameter of less than 10 mm. Genomic DNA was prepared from leukocytes of peripheral venous blood using the standard phenol/ chloroform method [22]. . Primers used to amplify the coding exons and adjacent intronic regions of the 6 genes were referred to a previous publication [23] with modification for a few primers (Table 1). Individual exon was amplified by polymerase chain reaction (PCR). The sequence of the amplicons was determined with the ABI BigDye Terminator cycle sequencing kit v3.1 on a genetic analyzer (ABI Applied Biosystems, Foster City, CA). Sequencing results from patients were aligned with consensus sequences to identify variations by using the SeqManII program of the Lasergene package (DNAStar Inc., Madison, WI). A variant detected in patient was further evaluated in controls by sequencing 96 normal individuals.

RESULTS
Upon complete analysis of the 6 genes, three heterozygous mutations in 2 genes were detected in 3 families (Figure 1), including c.34C>T (p.Arg12Cys) mutation in CRYAA, and c. 116C>G (p.Thr39Arg) and c.136G>A (p.Gly46Arg) mutations in GJA8, where the last two mutations are novel. Both of the c.116C>G and c.136G>A mutations in GJA8 are predicted to be "probably damaging" by PolyPhen-2 and "intolerant" by SIFT. The p.Thr39Arg would change the Blosum62 score from 4 to −1 whereas the p.Gly46Arg would change the Blosum62 score from 6 to −2. The p.Thr39Arg and p.Gly46Arg variants involved residues that are conserved across different species (Figure 2).
The heterozygous c.34C>T mutation in CRYAA was identified in all three patients in a three-generation family (QT597), where all patients had congenital nuclear cataract and microcornea (Figure 3, Table 2). Myopic fundus change in both eyes were observed in the affected father (II:1) and    The c.136G>A mutation in GJA8 was identified in a twogeneration family (QT204) with complete opacity of the lens and microcornea (Table 2). Horizontal cornea diameter was 9 mm for both eyes of the affected mother and 7 mm for both eyes of the affected daughter at the age of 5 years old.
The c.116C>G mutation in GJA8 was identified in a sporadic patient (QT895) of 7 years old with microcornea, complete opacity of lenses, and iris hypoplasia ( Figure 5, Table 2). Horizontal corneal diameter was about 6 mm for both eyes.

DISCUSSION
In this study, we screened 6 genes for mutations in 9 Chinese families with congenital cataract and microcornea. Three mutations were identified in 3 of the 9 (30%) families, including a c.34C>T (p.Arg12Cys) in CRYAA, and a c. 136G>A (p.Gly46Arg) and a c.116C>G (p.Thr39Arg) in GJA8, respectively.
CRYAA is located in 21q22.3 and encodes the α-Acrystallin in lens epithelial cells and fiber cells. α-A-crystallin is a member of small heat shock proteins with the chaperone activity which contributes to keeping lens transparent [6,10,27]. Up to now, there were eight mutations of CRYAA found in sixteen families most of which involved substitutions from or to arginine [5]. And the corresponding phenotypes of the mutations were related with congenital cataract with or without microcornea, microphthalmia, or iris coloboma.
We found a known c.34C>T (p.Arg12Cys) mutation in CRYAA of three patients from a family with congenital nuclear cataract and microcornea. Previously, this mutation has been identified in four families with nuclear or lamellar cataracts, and some patients accompanied with microcornea or microphthalmia [6,10,28,29]. Elongation of axial length or myopia has not been observed in previous studies.
GJA8 is located in chromosome 1q21.1 and encodes the gap junction proteins, connexin50. GJA8 is one of the most common genes causing congenital cataract with or without other ocular abnormalities. Previous studies showed that GJA8-knockout mice developed nuclear cataract and microphthalmia, from which it is considered that GJA8 plays a role not only in keeping lens transparent but in ocular growth [30,31]. Up to now, about twenty mutations in GJA8 have been associated with congenital cataracts in at least 21 families. Of these mutations, five were identified in five families with microcornea and two families accompanied with microphthamia [32,33].
In this study, we found two novel missense mutations c. 136G>A and c.116C>G in GJA8 in two families with congenital cataract and microcornea. The c.136G>A mutation led to a substitution from glycine to arginine at the amino acid position 46, and the c.116C>G mutation led to a substitution from threonine to arginine at the amino acid position 39. Both the 46 and 39 positions are located in the first transmembrane domain. In a previous study, Minogue et al. [32] identified a c.137G>T (p.Gly46Val) mutation in GJA8 of a proband with early-onset total cataract accompanied with small eyes and pupils. Therefore, the three mutations may result in phenotype by the similar mechanism.
In summary, a known c.34C>T mutation in CRYAA and two novel mutation in GJA8 were identified in 3 of 9 families after comprehensive analysis of 6 genes known to cause cataract and microcornea. Our results expand the mutation spectrum of GJA8 and phenotypic variations associated with CRYAA mutations. Patients without mutation in the 6 genes are potential candidate for future study of additional causative genes for cataracts and microcornea.